1. Field of the Invention
This invention relates to a method for the quantitation of percent glyeated protein in a biological sample suitable for use with a reflectance meter such as used in the self monitoring of blood glucose concentration by diabetics.
2. Description of the Related Art
Control of blood glucose concentrations in diabetics has been shown to decrease the frequency and severity of long-term microvascular and neurologic complications of the disease. The measurement of glycated hemoglobin and protein in blood are used to determine how well blood glucose concentration has been managed over an extended time period.
The rate of formation of glycated hemoglobin is directly related to the glucose concentration in blood.
The average red blood cell life span is 120 days, so quantitation of the percent glycation of hemoglobin has been correlated to a measure of the average glucose concentration over the previous 2 to 3 months which is a measure of glycemic control over that time period (see “Diabetes Control and Complications Trial Research Group, The effect of intensive treatment of diabetes on the development of progression of long-term complications in insulin-dependent diabetes mellitus”, New England Journal of Medicine, 329, 977-986 (1993), and “American Diabetes Association, Tests of Glycemia in Diabetes”, Diabetes Care, 20 (suppl. 1), S18-S20 (1997).
Glucose also attaches to non-hemoglobin proteins in blood, for example albumin. Since albumin is the most abundant serum protein and its circulating half-life is about 20 days, the concentration of glyeated protein is a measure of the average glucose concentration over the previous 2 to 3 weeks. The measure of glucose directly gives the glucose concentration at the time of measurement.
An immobilized dihydroxyboryl compound has been reported as useful to bind to the 1,2 cis diols of the carbohydrate of glycated proteins to separate them from non-glycated proteins. Use of this technology in a column chromatography method to determine percent glycation has been reported (see, U.S. Pat. No. 4,269,605 issued May 26, 1981 to Dean and U.S. Pat. No. 5,284,777, issued Feb. 8, 1994 to Rosenthal). These methods are said to use the boronate derivative immobilized onto agarose beads in a column to separate glycated from non-glycated proteins in the sample. These methods require specific dilutions and pipettings of the sample so as to not overload the capacity of the affinity binder affixed to the agarose beads. The use of a boronate derivative immobilized on agarose beads would not appear to lend itself to a strip application.
U.S. Pat. No. 5,110,745 issued May 5, 1992 to Kricka, et al., is said to describe methods of detecting glycated protein in a sample wherein the sample is contacted with a defined excess of a boronate compound in solution. The resulting unbound boronate is said to be measured by binding it to an immobilized glycated molecule on a support matrix and measuring the amount of glycated molecule left un-complexed. This method appears to require a number of steps including, a separate reaction in solution before application to a solid support, dilution of the sample to assure that the amount of binder added to the biological sample is in excess, performance of a separate assay to determine the percentage of protein glycation and multiple binding and washing steps.
A dipstick method for the measurement of glycated hemoglobin is said to be described in U.S. Pat. No. 4,861,728 issued Aug. 29, 1989 to Wagner. This method is said to involve contacting of a hemoglobin binding agent linked to a solid support with a lysed blood sample previously mixed with a dihydroxyboryl compound linked to a fluorescent label. The support is said to bind non-glycated hemoglobin and fluorescent labeled glycated hemoglobin. The fluorescent label is said to bind to glycated hemoglobin through the dihydroxyboronyl compound. The solid phase is removed from the sample and total hemoglobin is measured by reflectance photometry while glycated hemoglobin is measured using fluorescence. This method is said to require an addition of an amount of fluorescent labeled dihydroxyboryl reagent to the sample and a rinsing step after it is removed from the sample. Further it requires two different measurement methods for the quantitation.
In other assays, (see, e.g., Japanese Pat. No. 6,058,936, European Pat. No. 455225, and published PCT application WO 96/03657) a boronate derivative coupled to a detectable label (such as a fluorescent compound, a chemiluminescent compound, isotope, enzyme or other label) is said to be used. Both the glycated and non-glycated proteins are bound to a solid support using a general affinity binder such as an antibody. The boronate-label complex is added and the amount of label that remains bound to the solid support is measured. Each of these types of methods requires the additional step of labeling the glycated protein. In addition, these assays use different measurement methods to quantitate total and glycated proteins.
Published PCT application WO 9840750 (published Sep. 17, 1998) is said to describe a method of determining the percentage of glycated hemoglobin in which immobilized boronate binds glycated hemoglobin in the sample. The amount of glycated hemoglobin bound is said to be proportional to the fraction of glycated hemoglobin in the sample. This is said to eliminate the need for measuring nonglycated hemoglobin to determine the percent glycation. However, it appears the method results may vary depending on the incubation time of glyeated protein with the boronated support.
Consequently, there is a need for a simple, fast and efficient method to quantitate the amount of glycated protein in a biological sample that does not require dilution of the sample, requires minimal procedural steps, may be utilized in conjunction with a simple detection device such as a hand held reflectance meter, and is adaptable to a standard strip assay.